Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex.

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by N-linked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential N-linked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.

Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins.

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.

Characterization of truncated forms of hepatitis C virus glycoproteins.

Hepatitis C virus (HCV) glycoproteins (E1 and E2) both contain a carboxy-terminal hydrophobic region, which presumably serves as a membrane anchor. When they are expressed in animal cell cultures, these glycoproteins, in both mature complexes and misfolded aggregates, are retained in the endoplasmic reticulum. The effect of carboxy-terminal deletions on HCV glycoprotein secretion and folding was examined in this study. Sindbis and/or vaccinia virus recombinants expressing truncated forms of these glycoproteins ending at amino acids 311, 330, 354 and 360 (truncated E1), and 661, 688, 704 and 715 (truncated E2) were constructed. When expressed using Sindbis virus vectors, only truncated forms of E1 and E2 ending at amino acids 311 (E1t311) and 661 (E2t661), respectively, were efficiently secreted. Analysis of secretion of truncated forms of E2 glycoprotein expressed by vaccinia viruses indicated that significant secretion was still observed for a protein as large as E2t715. However, only secreted E2t661 appeared to be properly folded. Secreted HCV glycoprotein complexes were also detected in the supernatant of cell culture when E1t311 and E2t661 were coexpressed. Nevertheless, these secreted complexes, as well as E1t311 expressed alone, were misfolded. The effect of coexpression of E1 and E2 glycoproteins on each other's folding was evaluated with the help of a conformation-sensitive monoclonal antibody (for E2) or by analysing intramolecular disulfide bond formation (for E1). Our data indicate that the folding of E2 is independent of E1, but that E2 is required for the proper folding of E1.